The invention concerns methods and devices for isoelectric separation of particles whose charge characteristics depend on the pH value of a guiding fluid, especially for separating ampholytic suspended particles, colloids or biological cells. The invention concerns in particular the separation of such particles from a guiding fluid flow.
Numerous separation techniques are known from molecular biology; biochemistry, medicine and biotechnology whose function is based on charge differences of molecules within a substance that is to be separated. In the case of amphoteric ion compounds (socalled ampholytes or ampholytic molecules), the molecular charge depends on the pH value of the surrounding solution. The isoelectric point (hereafter referred to as the IP) of a compound is the pH value at which the net charge of the amphoteric molecules equals zero. The charge of the molecule is positive for a pH value smaller than the IP and negative in the opposite case.
In isoelectric focusing (hereafter referred to as IEF), proteins with different IPs are separated by making use of spatial pH gradients along a separation length (see R. A. M. Osher et al. in xe2x80x9cThe Dynamics of Electrophoresisxe2x80x9d, published by B. J. Radola, VCH, Weinheim, 1992, pp 163-231). IEF is performed, for example, using porous gel matrices or, for analytical separation of the smallest samples, using thin capillaries as what is called cIEF (see F. Foret et al. xe2x80x9cCapillary Electrophoresisxe2x80x9d in xe2x80x9cAdvances in Electrophoresisxe2x80x9d, vol. III, published by A. Chrambach et al., VCH, Weinheim, pp 273-347).
For the following reasons conventional IEF presents disadvantages and its use is restricted. Retrieval of the separated substances from a carrier material, especially for further processing like analysis, medication applications or the like, calls for elaborate procedures by which the separated substances themselves may be modified, or which lead to substance losses. The necessary use of additional substances to form a pH gradient that is as wide and linear as possible produces restrictions in terms of further use of the separated samples. The socalled carrier ampholytes used as additional substances are, in chemical terms, a highly diverse substance mix that is difficult to separate from the separated protein fractions. Furthermore, cIEF is restricted to minimal sample quantities that cannot be collected separately and are not separable from the carrier ampholytes.
The problem when socalled IPG membranes are used to form the pH gradient is that proteins, because of the necessity of passing through such a membrane, must pass through a milieu whose ionic concentration is very low. Consequently, sensitive proteins can denaturize or precipitate in this region. For this reason the strictest requirements are made in IEF for uniformity of the voltage gradients in the separation length. Finally, problems can occur in IEF in the form of electroosmosis in extreme pH ranges, the carrier medium (gel) heating up and altering (destruction of the gel) for instance.
To overcome such drawbacks, systems were developed in which ampholyte separation is produced by the effect of an external electric field and without modifying additional substances. One separating system is known, for example, that works by the method of socalled electric field flow fractionating (eFFF) (see K. D. Caldwell et al. in xe2x80x9cSciencexe2x80x9d, vol. 176, 1972, pp 296-298). In this separating system a continuous fluid stream is conducted through a narrow duct between two ion flow permeable membranes, into which the sample to be separated is injected as a narrow band. External electrodes, in electrical contact with the duct through a surrounding fluid, produce different degrees of retardation, as a function of charge, of the protein molecules in the fluid stream. However, use of this method is restricted to protein molecules with IPs that are relatively far apart. Furthermore, no complete protein separation could be achieved. Control of the pH value of the fluid streamxe2x80x94and thus of the molecular charge statexe2x80x94is not a facet of this familiar separating system.
The eFFF system is not applicable in practice. Although the samples could be separated (incompletely), collection of the separated fractions was not implemented. Furthermore, the times for separation are unacceptably high. A follow-on development of the above mentioned eFFF system (see L. F. Kesner et al. in xe2x80x9cAnalytical Chemistryxe2x80x9d, vol. 48, 1976, pp 1834-1839) produced analysis or separation times of several hours even on a laboratory scale for example.
A modified eFFF system described by Lightfoot et al. (see xe2x80x9cSeparation Science and Technologyxe2x80x9d, vol. 16, 1981, pp 619-656) makes use off cylindrical duct geometry. The separation performance of this system was also unacceptable in practical terms. Furthermore, protein retardation showed itself to be a complex function of a large number of parameters, eg the buffer ions that were used, the sample quantity, the sort of protein and properties of the duct wall. Nor is this familiar system intended for pH control or, say, separation of proteins with different IPs.
There is pronounced interest in substance separation for the production of high-purity substances, beyond the sphere of the laboratory, on an industrial scale. Because of the restrictions and disadvantages mentioned however, no continuous substance separation is known to date, using the named systems, that produces adequate separation performance and speed for the practical sphere.
The object of the invention is to indicate improved methods of isoelectric particle separation distinguished, in particular, by higher separation speed, greater reliability, and a broader range of application in terms of separable particles and the surrounding solutions. The object of the invention is also to indicate corresponding, continuous isoelectric purification methods. The object of the invention also consists in providing devices for implementing the methods of particle separation and further application possibilities.
The named objects are achieved by methods for isoelectric particle separation in which particles with a net charge or charge density that is a function of the pH value of the surroundings are exposed to electric field forces in a guiding fluid passing by collection means, whereby the pH value of the guiding fluid is set so that, through the effect of the electric field forces, at least one predetermined particle type undergoes a change in motion as a function of charge and is moved to the collection means, intended for soluble fixing of the charged particles or by devices for isolectric particle separation that include: electrode means for forming an electric field in a guiding fluid with particles whose net electric charge or charge density is a function of the pH value of the guiding fluid; pH setting means being adapted to set the pH value of the guiding fluid so that at least one predetermined particle type of the particles in the guiding fluid undergoes a change in motion as a function of charge through the effect of the electric field; and collection means arranged between the electrode means and the guiding fluid for soluble fixing of charged particles. Preferred embodiments of the invention are disclosed hereinafter.
The separation technique according to the invention is based on the idea of controlling or setting the pH value in a guiding fluid in which the particles to be separated are exposed to electric field forces so that all particle types with a net charge go through a motion dependent on charge, and the remaining, for the most part neutral particles show no change in their state of motion, whereby the particles moving as a function of charge are moved to collection means (collection device). The particles are retained at least temporarily on the collection means. This involves fixing on the surface or in the volume of the collection means. The duration of fixing is determined by the separation conditions, especially modulation with time of the electric field forces, alteration of the pH value, the structure of the collection means and/or relative motion between the collection means and the guiding fluid.
A major difference from the eFFF technique described above is thus that the particles are not differently retarded as a function of charge but that, through control of the pH value in the guiding fluid, a condition is created that defines what type of particle, exposed to the effect of an external electric field, moves into an at least temporarily fixed state and is possibly released again. To distinguish it from the eFFF technique, isoelectric separation according to the invention is therefore named pH-controlled electroretention chromatography. The collection means are effective as real restrain means. Thus a method is proposed for inverse isoelectric particle separation in which particles with a charge dependent on pH value are passed by collection means in a guiding fluid and the pH value of the guiding fluid is set or modified so that only the uncharged particles remain in the guiding fluid, while the charged particles are moved through an electric field to the collection means and are consequently fixed, at least temporarily, as a function of pH value.
The induced change of motion of the predetermined type of particle, according to the invention, is directed to the collection means so that the predetermined type of particle that is to be separated arranges itself or collects on or behind the collection means. The collection means are formed of a collection arrangement, for example, between electrode means to generate the external electric field and the guiding fluid. It is possible to move the collection means in relation to the guiding fluid.
The collection arrangement is preferably semi-permeable as a function of substance. Semi-permeability can mean, for instance, that the collection arrangement is permeable for the molecules of the guiding fluid or for ions dissolved in the guiding fluid, but impermeable, ie a barrier effect, for the substance or type of particle to be separated. Semi-permeability can also be implemented so that some of the type of particle to be separated with smaller particle sizes are let through, and the remainder of the particles to be separated with larger particle sizes are not let through. The collection arrangement is preferably a delimitation of the guiding fluid from the particles to be separated. Outside of the collection arrangement, in a surrounding solution with adjustable pH value, are the electrode means for forming the electric field.
The invention can be implemented with a static or a flowing guiding fluid. In both cases the collection arrangement forms a compartment or a vessel with at least one opening for entry or exit of the sample to be separated.
The separating effect is especially good and fast if the displacement path of the particles from the location of the sample in the guiding fluid (possibly flowing tangentially to the adjoining collection means) to the collection means is kept as small as possible. Preferably the collection arrangement will have characteristic dimensions that are in the flow direction of the guiding fluid much greater than the displacement path. The displacement path or inner dimensions of the compartments are of the order of mm or smaller for example.
In one preferred embodiment, the collection arrangement is formed of at least one longish, hollow element (eg tubular) that has semi-permeable or porous walls and through which the guiding fluid flows with the particle sample to be separated. Other forms of collection arrangement are also possible, especially with a rectangular instead of a round cross-section. In one particularly advantageous and preferred implementation the collection arrangement consists of at least one straight or bent hollow fiber with, at least in part, semi-permeable walls.
Isoelectric separation according to the invention can be implemented with ampholytic molecules or all other synthetic or biological particles (especially cells or viruses) that exhibit electrical characteristics like those of ampholytic molecules, in particular a net charge or charge density that is a pH function of the surroundings.
Optionally, in order to purify the guiding fluid, particles that are to be removed from the guiding fluid may be moved out of the guiding fluid by the collection means. The flow velocity of the guiding fluid may be reduced or dropped to zero at least temporarily when the particles to be separated are exposed to the electric field forces. A sample with the particles to be separated and/or the guiding fluid may be fed through microducts in the porous wall. The pH value of a sample solution, with which the particles are introduced to the guiding fluid, of the guiding fluid and/or of the surrounding solution may be set independently so that predetermined pH gradients form on the collection means. The pH value of a sample solution, in which the particles to be separated are introduced to the guiding fluid, and/or of the guiding fluid may be set by diffusive exchange processes with the surrounding solution.
Depending on the application, the purpose of isoelectric separation according to the invention can be aimed at obtaining the predetermined particle type that undergoes a change of motion in the electric field as a function of charge, or the remaining particles without change of the state of motion.
A device for isoelectric particle separation according to the invention contains electrode means for forming an electric field in a guiding fluid with the particles that are to be separated, and means for controlling or setting the pH value in the guiding fluid. There are also collection means between the electrode means and the guiding fluid that, in addition to a collecting function for a type of particle that is to be separated, also have a delimiting function from a surrounding solution with adjustable pH value. The collection means are permeable at least for ions dissolved in the surrounding solution and the guiding fluid, so that the pH value of the guiding fluid can also be set and controlled by setting the pH value of the surrounding solution.
Preferred forms of application of the invention are all separation processes on sample mixes that contain at least one particle type with ampholytic characteristics, and/or investigations of certain substances for determining the isoelectric point, of titration curves or aggregation response. The device according to the invention can also be used to reduce the ionic concentration of the guiding fluid in the field of pH-controlled electrodialysis.
The present invention provides also a device for isolectric particle separation that includes: electrode means for forming an electric field in a guiding fluid with particles whose net electric charge or charge density is a function of the pH value of the guiding fluid; pH setting means being adapted to set the pH value of the guiding fluid so that at least one predetermined particle type of the particles in the guiding fluid undergoes a change in motion as a function of charge through the effect of the electric field; and collection means arranged between the electrode means and the guiding fluid for soluble fixing of charged particles.
Isoelectric particle separation according to the invention possesses the following advantages:
Fractionating or separation occurs without additional substances, in particular without carrier ampholytes or other means such as for generating a pH gradient for instance. Since no pH gradient is generated, this simplifies considerably a preparative procedure in the flow-through system or collection of the required (possibly even all) separated fractions. The separated or purified fractions are available for further manipulation after separation, without having changed from the original form. The separated fractions are, in particular, always in the same solution with the same, possibly slightly reduced or even higher concentration. The system according to the invention can be used and automated for both discontinuous and continuous separation of particles as a function of their IPs. Both spatial fractionation and, by variation of the pH value during the separation process, fractionation as a function of time is possible. The speed of separation is high, of the order of minutes, as will be exemplified in what follows. The separation system allows configuration of the electrode means within tight confines so that, compared to conventional separation processes, sufficiently high field strength can be produced with relatively small voltages. This minimizes the energy consumption of systems according to the invention and relaxes conditions for samples sensitive to temperature.
Another aspect of the present invention is the use of a device for separation of at least one particle type from the guiding fluid to obtain a purified fraction of the separated particles and/or a purified guiding fluid, determining electrical and thermodynamic characteristics of substances like isoelectric point, titration curves or aggregation response, boosting the concentration of a particle solution or suspension, or reducing the ionic concentration of the guiding fluid.